PROJECT SUMMARY/ABSTRACT This proposal outlines a five-year career development program for Dr. Ansuman Satpathy, M.D., Ph.D. with the goal of preparing him for an independent research career as an academic physician-scientist. Dr. Satpathy completed his M.D. and Ph.D. in Immunology at Washington University in St. Louis and his medical residency in Clinical Pathology at Stanford Hospital and Clinics. During his residency, Dr. Satpathy performed basic science research in the lab of Dr. Howard Chang, who is a Professor of Dermatology and Director of the Center for Excellence in Genome Science at Stanford University. Dr. Chang is an expert in the fields of genomics and cancer biology and has previously served as a mentor to many physician-scientists who have transitioned to independent positions in academic medicine. Stanford University provides an outstanding environment for Dr. Satpathy to develop his independent research career. First, Dr. Satpathy's Advisory Committee comprised of Drs. Mackall, Montine, Greenleaf, and Galli has diverse and extensive scientific expertise relevant to all aspects of this proposal and highly successful track records as scientific mentors. Second, Dr. Satpathy will acquire additional scientific and professional skills through educational resources available through the School of Medicine and Office of Postdoctoral Affairs. Third, the research infrastructure within the Chang lab and neighboring core facilities will enable Dr. Satpathy to efficiently perform the scientific aims described in this proposal. The long-term goal of the proposed research is to study the epigenetic basis for immunotherapy resistance in patients with advanced basal cell carcinoma (BCC). Clinical immunotherapies that enhance the ability of T lymphocytes to destroy cancer cells have demonstrated remarkable efficacy in advanced skin cancers, but only a subset of patients respond to therapy. During Dr. Satpathy?s brief time in the Chang lab, he developed novel sequencing technologies which enable epigenomic studies in primary immune cells from patients with newfound resolution. In Aim 1, we will perform simultaneous epigenome and T cell receptor sequencing in single cells to identify epigenetic signatures of T cell dysfunction in tumor-specific CD8+ tumor- infiltrating T lymphocytes (TILs). This integrative approach will separate regulatory networks in clonal tumor- specific T cells from background non-reactive T cells in the same patient. In Aim 2, we will use this technique to identify regulatory signatures in CD8+ TILs that are associated with immunotherapy resistance in BCC patients receiving anti-PD-1 immunotherapy. In Aim 3, we will engineer durable T cell immunotherapy responses using genome editing of regulatory sites in primary T cells. Finally, we will facilitate the dissemination of these findings by freely distributing protocols and data and releasing custom software tools. We anticipate that these results will lead to novel insights into the molecular regulation of immunotherapy resistance and serve as an effective training program for Dr. Satpathy to launch his independent career as an academic physician scientist.